LIPID GLOBULES AND FATTY ACIDS IN MILK OF LACTATING RACCOON ( NYCTEREUTES PROCYONOIDES )
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ANIMAL S CIENCE S
Lipid Globules and Fatty Acids in Milk
of Lactating Raccoon (Nyctereutes
procyonoides)
O. Szeleszczuk1, P. Kilar1, D. Maj2
1 Universityof Agriculture in Krakow, Department of Animal Reproduction, Anatomy and
Genomics, Krakow, Poland
2University of Agriculture in Krakow, Department of Genetics, Animal Breeding and
Ethology, Krakow, Poland
In this study, milk samples (n=52) from nine healthy female raccoons at 3–45 days of lactation were manually collected at a
regular morning hour from all active teats and used to investigate the changes of lipid globules size, fatty acids (FAs) profile
and fat content.The results indicated that raccoon milk is characterized by a high fat content. Small lipid globules sizing up to
6 µm prevailed with their greatest share in milk during lactation phases I and III. The milk fat content was increasing with the
proceeding lactation, whereas the content of free FAs had a decreasing tendency. Totally eighteen FAs were identified in rac-
coon milk. The unsaturated long-chain C18–C20 FAs were dominating (over 60%). The individual FAs contents in raccoon
milk did not exceed 1%, except for palmitic, vaccenic and linoleic acids representing over 20% of the total FA content.The
study results can be used for establishing the energy requirements during the suckling period for proper growth and develop-
ment of puppies.
carnivora,fatty acids, milk fat, Nyctereutes procyonoides, raccoon milk
doi: 10.2478/sab-2021-0002
Received for publication on July 22, 2020
Accepted for publication on February 10, 2021
Introduction higher than the total of dead animals raised during the
whole breeding cycle. Losses in fox offspring have
Mammals are the only group of animals with a still been a problem (H a n s e n , 2008) visible also
mammary gland with the evolved mechanism of milk in the other species of fur animals, both herbivorous
production. Secretion produced in the gland is the only and carnivorous. According to S z e l e s z c z u k et
source of food for the offspring in the first weeks after al.(2008), this phenomenon can also be observed in
birth.In most mammals, milk is excreted only in the another species belonging to carnivorous fur animals –
period of lactation and contains nutrients necessary for the raccoon (Nyctereutes procyonoides). As indicated
proper functioning of young organisms in the first days by S z e l e s z c z u k et al.(2015), the perinatal period
of life. In mono-estrus breeding females of Canidae, and the first week of pups’ life are the most crucial for
lactation takes place only once a year and this process raccoon dogs. In that period, 30–48% offspring mor-
lasts for 6–8 weeks. Malfunctioning mammary glands tality has been recorded and the only food consumed
cause disorders in milk production and secretion which by pups is the milk. Female milk is decisive directly
can be a persistent problem in feeding the pups. The upon puppies’ proper growth and development. Milk
research results of S z e l e s z c z u k et al. (2007) in- lipids are their main energy source. Milk fat is diffused
dicate a current problem of mortality rate increase in in the water phase of milk in the form of small lipid
fox litters. The number of still-born and dead suckling globules stabilised due to their coating (E l - Z e i n i ,
kids in the first days after parturition is significantly 2006). The globules diameter is 0.1–15 µm. They can
Scientia agriculturae bohemica, 52, 2021 (1): 11–17 11be classified into three size categories: small (≤6 µm), were collected from all active milk glands of the fe-
medium (7–10 µm) and large (≥10 µm). Milk fat of males during a diagnostic examination by a veteri-
mammals consists of simple and complex lipids, free narian.The milk samples were taken at regular hours
fatty acids (FAs) and accompanying substances. The before early morning feeding. Before milking, the
simple lipids – triglycerides – have the biggest share milk gland health condition was examined. Its consist-
in the total milk fat (96–99%). They are esters com- ency, soreness or temperature was inspected manually.
posed of three molecules of FAs and one molecule of Blood or pus secretion was checked at each teat. Milk
glycerol (J e n s e n et al., 1991). intended for analysis was collected exclusivelyf rom
FAs are one of the most important substances deter- healthy raccoon females with healthy milk glands.
mining the development of both humans and animals Each female was subcutaneously injected at the scruff
in prenatal and postnatal periods. These compounds of the neck with 0.2 IU of oxytocin (Oxytocinum
are responsible for cell energetic processes and rep- Biowet, Poland) using a 2.54 cm 25-gauge needle. After
resent the main building element of cytomembranes 10 min to allow the hormones work, the teats were mas-
(B o b i n s k i , B o b i n s k a , 2020). saged.Milk for the assay was collected manually from
One of the factors affecting milk lipids is the lactation each active teat to 40 ml sterile plastic containers and
stage (M e s i l a t i - S t a h y , A r g o v - A r g a m a n , 2 ml Eppendorf tubes. The milk glands were tried to be
2014). During lactation, significant changes can be emptied completely to obtain the identical volume of
observed in milkfat and the FA content. Studies in milk as during the pups feeding.For the fat analysis,the
different mammals as well as humans have been con- collected milk was preserved by Broad Spectrum
ducted (J i a n g , 2020). However, little information on Microtabs II (dispenser with 800 tablets) (Advanced
lipids in Canidae milk and its changes during lactation Instruments, USA) (B a r l o w s k a , 2007). Milk sam-
(S z e l e s z c z u k et al.,2017) has been available in ples were transported to the laboratory in a cooled bag
the subject literature to date. Therefore the purpose and stored deep frozen(–20°C) until analyses.
of the study was to determine the fat content, the size
and number of lipid globules, and the FAs profile inr Laboratory analyses
accoon (Nyctereutes procyonoides) milk at various
stages of lactation. The fat and free FAs contents in raccoon milk
samples were determined using a MilkoScan FT2
analyser (FOSS, Denmark) calibrated by laboratory
Material and methods using cow milk samples. The FAs profile was deter-
mined according to F o l c h et al.(1957) using a gas
Animals chromatograph Varian 450-GC with a flame ioniza-
tion detector (FID) and a CP-SIL 88 column (length
Animals were handled following the guidelines for 100 m, diameter 0.25 mm) for FAME (all Agilent
animal experiments set in the D i r e c t i v e 2 0 1 0 / 6 3 / Technologies, USA). The injector temperature was
E U regarding the protection of animals used for ex- 220°C and the detector temperature 250°C. The car-
periments, and in accord with the permission from the rier gas was helium (1 ml min –1 ).The milk samples
II Local Ethics Committee in Krakow No. 17/2010 were extracted using chloroform and methanol mixed
and No. 1/2020. 2:1. Then, 0.58% NaCl was added, the samples were
Nine lactating female raccoons, 2–3 years old,were centrifuged, 1 cm 3 of the bottom layer was collected
used in this study. Before the reproductive season, and the solvent was evaporated under nitrogen (at
females were subjected to health selection and pre- 50°C). Totally 0.5M NaOH in methanol, 10% BF 3
ventive procedures (deworming and protective vac- in methanol and heptane were added respectively to
cinations). The animals were fed in accordance with the dry residue. After adding each reagent, the test
the feeding standards recommended for particular tube was screwed and put into a water bath (73°C).
lactation periods for this species (N R C , 1982) and Then, a saturated solution of NaCl was added. After
fresh water was available ad libitum. The females separating the layers, the heptanoic layer was re-
in lactation were fed three times daily. Each female moved and placed in a test tube containing anhydrous
nursed on average 6 puppies. Na 2 SO 4 . Individual FA methyl esters were identified
by comparison to the standard mixture (Supelco
Milk sampling 37 component FAME Mix; Sigma-Aldrich Co., USA).
The FA content was expressed as a percentage of the
The lactation period was divided into three phases: total identified FAs.
phase I (early): up to 15 days; phase II (middle): from The size and amount of lipid globules were deter-
16 to 30 days; and phase III (later): from 31 to 45 days. mined in the samples of unpreserved raccoon milk
Totally 52 milk samples (phase I, n = 16; phase II, according to the method worked out and later modi-
n = 18; phase III, n = 18) were collected in days 3–45 of fied by B a r l o w s k a (2007). The performed slides
lactation at weekly intervals. The samples (10–32 ml) were examined under a microscope Nikon ECLIPSE
12 Scientia agriculturae bohemica, 52, 2021 (1): 11–17Table 1.Quantity (ml) and proportion of lipid globules (%) of raccoon milk depending on lactation phase
Phase of lactation
Items
I II III
Milk quantity (ml) 7.00 a (7.00–10.00) 22.00b (17.00–27.00) 17.00b (7.00–22.00)
Lipid globules proportion (%)
< 6 µm 85.34a (84.77–89.73) 75.00 a (68.75–84.28) 83.46 a (81.45–93.48)
7–10µm 13.61 a (9.73–15.23) 15.24 a (13.27–25.00) 12.40 a (6.05–14.97)
> 10µm 0.54 a (0.39–1.05) 0.48 a (0–1.56) 2.97 a (0.47–4.30)
values are presented as median and interquartile range (Q1= lower quartile, Q3= upper quartile)
a,bwithin a row, means with a common superscript do not differ significantly (PDiscussion In our experiment, the amount of milk collected
from the female raccoons (2–32 ml per dam) was lower
The information on the raccoon milk lipid contentis in comparison to that yielded by other farm animals.
missing in available literature. Therefore, the present Foxes, belonging also to the family Canidae,yielded
research results were compared with results available 2–300 ml milk per dam per day 2–3 days post partum,
on other carnivores such as Canidae and Felidae. and the yield per dam increased to 7–800 ml in days
13–16 post partum. The maximum daily milk yield ob-
served in a silver fox dam nursing eight 16-day-old cubs
Table 3.Fatty acids profilein raccoon milk (%of total fatty acids) was estimated at 858 ml (A h l s t r o m , W a m b e r g ,
2000). It is not easy to compare data from different
Fatty acids Percentage of total FA
studies including the milk yield data, because many of
C4:0 butyric 0.05 ± 0.02 the results are influenced by species, animal feeding
C6:0 caproic 0.25 ± 0.11 and stage of lactation.
C8:0 caprylic 0.01 ± 0.00 In the raccoon milks mear performed within this
C10:0 capric 0.04 ± 0.02 study, small lipid globules sizing up to 6 µm pre-
C12:0 lauric 0.52 ± 0.36
vailed while globules over 10 µm were less frequent.
Similar trends were also observed in silver foxes
C14:0 myristic 1.67 ± 0.31
(S z e l e s z c z u k et al.,2017). According to S i n g h
C15:0 pentadecanoic 0.13 ± 0.04 (2019) the occurrence of smaller globules is more
C16:0 palmitic 23.93 ± 1.11 beneficial as they have a larger surface for a contact
C16:1n7 palmitoleic 4.67 ± 0.44 with enzymes that decompose fat, whereby they are
C18:0 stearic 4.99 ± 0.49 more easily digestible for the organism. Similarly,
M a r t i n i et al. (2012) stated that smaller lipid glob-
C18:1n9 cis oleic 0.20 ± 0.10
ules are more available and susceptible to lipolytic
C18:1n7 trans vaccenic 38.13 ± 2.28
processes. Moreover, small lipid globules contain more
C18:2n6 linoleic 20.27 ± 1.48 short-chain FAs and less long-chain ones as opposed
conjugated to greater globules. L u et al. (2016) found that fat
C18:2 CLA 0.08 ± 0.09
linoleic acid globules show species variability. Many studies on
C18:3n3 linolenic 1.60 ± 0.35 fat globules in ruminant milks have been conducted,
C20:0 arachidic 0.05 ± 0.01 unfortunately, there is little information about fat
globules in carnivore milk in scientific literature,
C20:1n9 eicosenoic 0.38 ± 0.17
making the comparison difficult.
C20:2n6 eicosadienoic 0.25 ± 0.02 In our study the detected fat content in raccoon milk
Remaining acids was high (9.25–13.61%). Similar results have been
3.05
(altogether) reported for the milk of silver and blue fox females
values are presented as mean ± SD
Table 4.Saturated fatty acids profile (%) of raccoon milk in relation to lactation phase
Phase of lactation
Saturated fatty acids I
II III
C4:0 0.06a ± 0.01 0.04a ± 0.02 0.05a ± 0.01
C6:0 0.45a ± 0.05 0.30 b ± 0.16 0.22b ± 0.04
Short chain FA C8:0 0.02a ± 0.01 0.01 b ± 0.00 0.01b ± 0.01
C10:0 0.06a ± 0.03 0.04ab ± 0.02 0.04 b ± 0.02
total 0.53 0.35 0.37
C12:0 0.85 a ± 0.02 0.86a ± 0.24 0.24c ± 0.01
C14:0 2.49a ± 0.41 1.93 b ± 0.28 1.46c ± 0.12
C15:0 0.18a ± 0.04 0.13a ± 0.05 0.12a ± 0.04
Long chain FA C16:0 24.20a ± 0.84 25.03a ± 0.29 23.00 b ± 0.39
C18:0 4.97 ab ± 0.22 5.34a ± 0.54 4.70 b ± 0.14
C20:0 0.04 ab ± 0.01 0.04a ± 0.01 0.05b ± 0.01
total 32.73 33.33 29.57
values are presented as mean ± SD
a–cwithin a row, means with a common superscript do not differ significantly (PTable 5. Unsaturated fatty acids profile (%) of raccoon milk in relation to lactation phase
Phase of lactation
Unsaturated fatty acids I
II III
C16:1n7 4.27a ± 0.43 4.42 a ± 0.41 4.88 a ± 0.37
C18:1n9cis 0.33a ± 0.15 0.23ab ±0.12 0.17 b ± 0.08
MUFA C18:1n7trans 38.48ab ± 0.53 36.59a ± 2.63 39.42b ± 0.70
C20:1n9 0.72 a ± 0.27 0.52b ± 0.07 0.25 c ± 0.11
total 43.80 41.76 44.72
C18:2n6 18.00a ± 0.64 18.82 a ± 0.64 21.48b ± 0.46
C18:2 CLA 0.04a ± 0.03 0.13 b ± 0.11 0.03 a ± 0.02
PUFA C18:3n3 1.50a ±0.50 1.32 a ± 0.14 1.84 a ± 0.29
C20:2n6 0.34a ± 0.04 0.27b ± 0.02 0.24 b ± 0.01
total 19.88 20.54 23.59
MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids
values are presented as mean ± SD
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